CN109232238A - Gemfibrozil Capsules haptens, artificial antigen and antibody and its preparation method and application - Google Patents
Gemfibrozil Capsules haptens, artificial antigen and antibody and its preparation method and application Download PDFInfo
- Publication number
- CN109232238A CN109232238A CN201810991947.1A CN201810991947A CN109232238A CN 109232238 A CN109232238 A CN 109232238A CN 201810991947 A CN201810991947 A CN 201810991947A CN 109232238 A CN109232238 A CN 109232238A
- Authority
- CN
- China
- Prior art keywords
- gemfibrozil capsules
- haptens
- gemfibrozil
- capsules
- antibody
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 229960003627 gemfibrozil Drugs 0.000 title claims abstract description 103
- HEMJJKBWTPKOJG-UHFFFAOYSA-N Gemfibrozil Chemical compound CC1=CC=C(C)C(OCCCC(C)(C)C(O)=O)=C1 HEMJJKBWTPKOJG-UHFFFAOYSA-N 0.000 title claims abstract description 102
- 239000002775 capsule Substances 0.000 title claims abstract description 102
- 239000000427 antigen Substances 0.000 title claims abstract description 46
- 102000036639 antigens Human genes 0.000 title claims abstract description 46
- 108091007433 antigens Proteins 0.000 title claims abstract description 46
- 238000002360 preparation method Methods 0.000 title claims abstract description 22
- 239000007788 liquid Substances 0.000 claims description 32
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 18
- 238000001514 detection method Methods 0.000 claims description 16
- 238000006243 chemical reaction Methods 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 238000004458 analytical method Methods 0.000 claims description 8
- 102000014914 Carrier Proteins Human genes 0.000 claims description 7
- 108010078791 Carrier Proteins Proteins 0.000 claims description 7
- 241001465754 Metazoa Species 0.000 claims description 7
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 229940098773 bovine serum albumin Drugs 0.000 claims description 6
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 6
- JODRRPJMQDFCBJ-UHFFFAOYSA-N 2-Hydroxy-4-methylbenzaldehyde Chemical compound CC1=CC=C(C=O)C(O)=C1 JODRRPJMQDFCBJ-UHFFFAOYSA-N 0.000 claims description 4
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 3
- 238000005352 clarification Methods 0.000 claims description 3
- 238000000502 dialysis Methods 0.000 claims description 3
- 238000004090 dissolution Methods 0.000 claims description 3
- 235000019441 ethanol Nutrition 0.000 claims description 3
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims description 3
- 238000000605 extraction Methods 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 3
- 230000028993 immune response Effects 0.000 claims description 3
- 239000012074 organic phase Substances 0.000 claims description 3
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 3
- 239000002994 raw material Substances 0.000 claims description 3
- 239000000741 silica gel Substances 0.000 claims description 3
- 229910002027 silica gel Inorganic materials 0.000 claims description 3
- 229960001866 silicon dioxide Drugs 0.000 claims description 3
- 229910052799 carbon Inorganic materials 0.000 claims description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims 1
- FUZZWVXGSFPDMH-UHFFFAOYSA-N hexanoic acid Chemical compound CCCCCC(O)=O FUZZWVXGSFPDMH-UHFFFAOYSA-N 0.000 claims 1
- 229910052739 hydrogen Inorganic materials 0.000 claims 1
- 239000001257 hydrogen Substances 0.000 claims 1
- 125000004435 hydrogen atom Chemical class [H]* 0.000 claims 1
- 229910052708 sodium Inorganic materials 0.000 claims 1
- 239000011734 sodium Substances 0.000 claims 1
- 238000003756 stirring Methods 0.000 claims 1
- 238000012360 testing method Methods 0.000 abstract description 12
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 238000000034 method Methods 0.000 description 25
- 239000000523 sample Substances 0.000 description 21
- 238000002965 ELISA Methods 0.000 description 17
- 230000015572 biosynthetic process Effects 0.000 description 11
- 239000000243 solution Substances 0.000 description 11
- 238000003786 synthesis reaction Methods 0.000 description 11
- 241000699666 Mus <mouse, genus> Species 0.000 description 9
- 238000002835 absorbance Methods 0.000 description 8
- 239000011248 coating agent Substances 0.000 description 8
- 238000000576 coating method Methods 0.000 description 8
- 229940092253 ovalbumin Drugs 0.000 description 7
- 241001494479 Pecora Species 0.000 description 6
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 6
- 238000011084 recovery Methods 0.000 description 6
- 239000007790 solid phase Substances 0.000 description 6
- 238000010790 dilution Methods 0.000 description 5
- 239000012895 dilution Substances 0.000 description 5
- 239000012224 working solution Substances 0.000 description 5
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- 108010058846 Ovalbumin Proteins 0.000 description 4
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 239000008363 phosphate buffer Substances 0.000 description 4
- 238000004321 preservation Methods 0.000 description 4
- 238000002203 pretreatment Methods 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 3
- 238000005138 cryopreservation Methods 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 210000004408 hybridoma Anatomy 0.000 description 3
- 230000001900 immune effect Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 230000010355 oscillation Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000012488 sample solution Substances 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- KZAMQAAMEOXHBU-UHFFFAOYSA-N 5-bromo-2,2-dimethylpentanoic acid Chemical compound OC(=O)C(C)(C)CCCBr KZAMQAAMEOXHBU-UHFFFAOYSA-N 0.000 description 2
- 206010003445 Ascites Diseases 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 230000005875 antibody response Effects 0.000 description 2
- 230000000890 antigenic effect Effects 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 239000005018 casein Substances 0.000 description 2
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 2
- 235000021240 caseins Nutrition 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 230000002860 competitive effect Effects 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 239000003547 immunosorbent Substances 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 230000031700 light absorption Effects 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 2
- 235000017557 sodium bicarbonate Nutrition 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000010189 synthetic method Methods 0.000 description 2
- OJRHUICOVVSGSY-RXMQYKEDSA-N (2s)-2-chloro-3-methylbutan-1-ol Chemical compound CC(C)[C@H](Cl)CO OJRHUICOVVSGSY-RXMQYKEDSA-N 0.000 description 1
- ZGGHKIMDNBDHJB-NRFPMOEYSA-M (3R,5S)-fluvastatin sodium Chemical compound [Na+].C12=CC=CC=C2N(C(C)C)C(\C=C\[C@@H](O)C[C@@H](O)CC([O-])=O)=C1C1=CC=C(F)C=C1 ZGGHKIMDNBDHJB-NRFPMOEYSA-M 0.000 description 1
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 description 1
- 239000005977 Ethylene Substances 0.000 description 1
- 208000031226 Hyperlipidaemia Diseases 0.000 description 1
- PCZOHLXUXFIOCF-UHFFFAOYSA-N Monacolin X Natural products C12C(OC(=O)C(C)CC)CC(C)C=C2C=CC(C)C1CCC1CC(O)CC(=O)O1 PCZOHLXUXFIOCF-UHFFFAOYSA-N 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- SUAUILGSCPYJCS-UHFFFAOYSA-N Musk ambrette Chemical compound COC1=C([N+]([O-])=O)C(C)=C([N+]([O-])=O)C=C1C(C)(C)C SUAUILGSCPYJCS-UHFFFAOYSA-N 0.000 description 1
- 239000005662 Paraffin oil Substances 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- RYMZZMVNJRMUDD-UHFFFAOYSA-N SJ000286063 Natural products C12C(OC(=O)C(C)(C)CC)CC(C)C=C2C=CC(C)C1CCC1CC(O)CC(=O)O1 RYMZZMVNJRMUDD-UHFFFAOYSA-N 0.000 description 1
- AJLFOPYRIVGYMJ-UHFFFAOYSA-N SJ000287055 Natural products C12C(OC(=O)C(C)CC)CCC=C2C=CC(C)C1CCC1CC(O)CC(=O)O1 AJLFOPYRIVGYMJ-UHFFFAOYSA-N 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- 239000004411 aluminium Substances 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical class N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 239000000730 antalgic agent Substances 0.000 description 1
- 238000003452 antibody preparation method Methods 0.000 description 1
- 229960001770 atorvastatin calcium Drugs 0.000 description 1
- HFACYLZERDEVSX-UHFFFAOYSA-N benzidine Chemical compound C1=CC(N)=CC=C1C1=CC=C(N)C=C1 HFACYLZERDEVSX-UHFFFAOYSA-N 0.000 description 1
- IIBYAHWJQTYFKB-UHFFFAOYSA-N bezafibrate Chemical compound C1=CC(OC(C)(C)C(O)=O)=CC=C1CCNC(=O)C1=CC=C(Cl)C=C1 IIBYAHWJQTYFKB-UHFFFAOYSA-N 0.000 description 1
- 229960000516 bezafibrate Drugs 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- KNHUKKLJHYUCFP-UHFFFAOYSA-N clofibrate Chemical compound CCOC(=O)C(C)(C)OC1=CC=C(Cl)C=C1 KNHUKKLJHYUCFP-UHFFFAOYSA-N 0.000 description 1
- 229960001214 clofibrate Drugs 0.000 description 1
- 239000003433 contraceptive agent Substances 0.000 description 1
- 230000002254 contraceptive effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 229960000868 fluvastatin sodium Drugs 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 230000000855 fungicidal effect Effects 0.000 description 1
- 239000000417 fungicide Substances 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 239000000118 hair dye Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 229940027941 immunoglobulin g Drugs 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 235000015110 jellies Nutrition 0.000 description 1
- 239000008274 jelly Substances 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- PCZOHLXUXFIOCF-BXMDZJJMSA-N lovastatin Chemical compound C([C@H]1[C@@H](C)C=CC2=C[C@H](C)C[C@@H]([C@H]12)OC(=O)[C@@H](C)CC)C[C@@H]1C[C@@H](O)CC(=O)O1 PCZOHLXUXFIOCF-BXMDZJJMSA-N 0.000 description 1
- 229960004844 lovastatin Drugs 0.000 description 1
- QLJODMDSTUBWDW-UHFFFAOYSA-N lovastatin hydroxy acid Natural products C1=CC(C)C(CCC(O)CC(O)CC(O)=O)C2C(OC(=O)C(C)CC)CC(C)C=C21 QLJODMDSTUBWDW-UHFFFAOYSA-N 0.000 description 1
- RLSSMJSEOOYNOY-UHFFFAOYSA-N m-cresol Chemical compound CC1=CC=CC(O)=C1 RLSSMJSEOOYNOY-UHFFFAOYSA-N 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- AJLFOPYRIVGYMJ-INTXDZFKSA-N mevastatin Chemical compound C([C@H]1[C@@H](C)C=CC2=CCC[C@@H]([C@H]12)OC(=O)[C@@H](C)CC)C[C@@H]1C[C@@H](O)CC(=O)O1 AJLFOPYRIVGYMJ-INTXDZFKSA-N 0.000 description 1
- 229950009116 mevastatin Drugs 0.000 description 1
- BOZILQFLQYBIIY-UHFFFAOYSA-N mevastatin hydroxy acid Natural products C1=CC(C)C(CCC(O)CC(O)CC(O)=O)C2C(OC(=O)C(C)CC)CCC=C21 BOZILQFLQYBIIY-UHFFFAOYSA-N 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229920006389 polyphenyl polymer Polymers 0.000 description 1
- TUZYXOIXSAXUGO-PZAWKZKUSA-N pravastatin Chemical compound C1=C[C@H](C)[C@H](CC[C@@H](O)C[C@@H](O)CC(O)=O)[C@H]2[C@@H](OC(=O)[C@@H](C)CC)C[C@H](O)C=C21 TUZYXOIXSAXUGO-PZAWKZKUSA-N 0.000 description 1
- 229960001495 pravastatin sodium Drugs 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000010865 sewage Substances 0.000 description 1
- 229960002855 simvastatin Drugs 0.000 description 1
- RYMZZMVNJRMUDD-HGQWONQESA-N simvastatin Chemical compound C([C@H]1[C@@H](C)C=CC2=C[C@H](C)C[C@@H]([C@H]12)OC(=O)C(C)(C)CC)C[C@@H]1C[C@@H](O)CC(=O)O1 RYMZZMVNJRMUDD-HGQWONQESA-N 0.000 description 1
- -1 small molecule compound Chemical class 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 125000003698 tetramethyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 238000011282 treatment Methods 0.000 description 1
- AQLJVWUFPCUVLO-UHFFFAOYSA-N urea hydrogen peroxide Chemical compound OO.NC(N)=O AQLJVWUFPCUVLO-UHFFFAOYSA-N 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C59/00—Compounds having carboxyl groups bound to acyclic carbon atoms and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
- C07C59/40—Unsaturated compounds
- C07C59/74—Unsaturated compounds containing —CHO groups
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/77—Ovalbumin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Urology & Nephrology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Hematology (AREA)
- Biotechnology (AREA)
- Gastroenterology & Hepatology (AREA)
- Pathology (AREA)
- Cell Biology (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Microbiology (AREA)
- General Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention provides a kind of Gemfibrozil Capsules haptens and artificial antigen, molecular structural formula to be respectively as follows:The invention also discloses this haptens, artificial antigen and with the preparation method of the antibody of this antigen preparation and the purposes of haptens, antibody.Antibody test high sensitivity that the present invention is finally made, high specificity.
Description
Technical field
The present invention relates to select it is a kind of have-CHO, maximum possible includes the compound of Gemfibrozil Capsules original structure again
As Gemfibrozil Capsules haptens, with artificial antigen made of this haptens, specific antibody；And it is such haptens, artificial anti-
The former, purposes of specific antibody and haptens, artificial antigen, antibody preparation method.
Background technique
The necessity of drug and personal care articles (PPCPs) as the mankind, broad categories, including it is all kinds of antibiotic, artificial
Musk ambrette, anodyne, depressor, contraceptive, somnifacient, slimming drugs, hair jelly, hair dye and fungicide etc..Gemfibrozil Capsules
It (GEM) is a kind of regulating plasma lipid drug, for treating the diseases such as hyperlipidemia.GEM cannot be gone completely after entering sewage treatment plant
It removes, as water outlet can enter in water environment, causes secondary pollution.Since its concentration level in surrounding medium is lower
(ng/L~μ g/L), therefore often ignored by people.Although the half-life period of PPCPs is shorter, endlessly inputted due to having
Source, causes it that the state of " pseudo- persistently to exist " is presented in surrounding medium, and long-term exposure causes to dive to biology and human health
It is endangering.With the enhancing of people's environmental protection consciousness, Gemfibrozil Capsules etc. " emerging pollutant " come in recent years increasingly by
Extensive concern, and establishing a series of detection method fast, at low cost, that accuracy is high of speed seems extremely urgent.
It is mainly at present instrumental method, such as liquid chromatography, liquid chromatography-mass spectrometry to the detection of Gemfibrozil Capsules.Instrument
Device detection method there are sample pre-treatments it is cumbersome, detection time is long, instrument is valuable the disadvantages of, answered extensively so being unable to get in China
With, and do not meet the requirement of on-site test " low cost is accurately detected and screened to a large amount of samples in a short time ".Enzyme
Linked immunosorbent assay technology Enzyme-Linked Immunosorbent Assay (ELISA) is by immunological technique and modern times
Means of testing combines and the measuring method of a kind of ultramicron established, has that at low cost, speed is fast, high sensitivity, instrument are set
Standby simple feature, is suitble to the quick analysis of batch samples.And the basis of immunological detection method be corresponding high specific,
The preparation of the antibody of high-affinity, and Gemfibrozil Capsules do not have immunogenicity in itself as small molecule compound, therefore, half
Structure of modification and the holoantigen synthesis of antigen are the key that establish immune one of quick test technique and difficult point.
Summary of the invention
For the shortcomings of the prior art, the present invention provides a kind of change that can utmostly retain Gemfibrozil Capsules
Learn structure, and the preparation method of the haptens with certain length linking arm and this haptens；With the preparation of this haptens
Artificial antigen, detection sensitivity be high and the antibody of high specificity；And the purposes of this haptens, antibody.
The present invention is technical solution in this way to realize to achieve the above objectives: providing a kind of Gemfibrozil Capsules half
Antigen, its molecular structural formula are as follows:
Haptens of the invention is anti-with bromo- 2, the 2- dimethyl valeric acid of 5- using 2- hydroxy-4-methyl benzaldehyde as starting material
Aldehyde radical Gemfibrozil Capsules, as Gemfibrozil Capsules haptens should be generated.Reaction equation is as follows:
The preparation method of Gemfibrozil Capsules haptens of the invention is further described below:
2- hydroxy-4-methyl benzaldehyde 0.5g is taken, acetonitrile 50mL is added to dissolve, clarification adds potassium carbonate 0.61g, adds 5- bromo- 2,
2- dimethyl valeric acid 0.84g, oil bath heating back flow reaction 3h；Stop reaction, be cooled to room temperature, rotate, removes acetonitrile, add water
50mL, ethyl acetate 100mL × 3, extraction three times, merge organic phase, upper silicagel column, with the ethyl acetate-pet of volume ratio 1:3
Gemfibrozil Capsules haptens can be obtained in ether chromatographic purifying.
Gemfibrozil Capsules haptens made from the above method, having again while having Gemfibrozil Capsules feature structure can be with egg
White matter coupling-CHO structure.
The present invention also provides a kind of by Gemfibrozil Capsules artificial antigen made from above-mentioned Gemfibrozil Capsules haptens, its point
Subformula are as follows:
The carrier protein is bovine serum albumin(BSA).
The preparation method of Gemfibrozil Capsules artificial antigen of the invention is described as follows:
Gemfibrozil Capsules haptens 9.8mg is taken, adds ethyl alcohol 0.2mL to dissolve, obtains A liquid；Carrier protein 50mg is taken, is added
0.1mol/L sodium bicarbonate solution 3mL dissolution, obtains B liquid；A drop is added in B liquid, 4 DEG C are stirred for 24 hours, normal saline dialysis
3d is purified, is changed daily liquid 3 times, is dispensed, -20 DEG C save backup.
Invention also provides the purposes of above-mentioned Gemfibrozil Capsules haptens, are used as the original of the antigen system of animal immune
Material.
Invention also provides above-mentioned Gemfibrozil Capsules artificial antigen, immune white mouse is obtained, can occur with Gemfibrozil Capsules
The Gemfibrozil Capsules monoclonal antibody of specific immune response, and its application in detection Gemfibrozil Capsules residual.
It may be its active group based on Gemfibrozil Capsules-COOH linking arm, to the immunological characteristic and feature for maintaining haptens
Structure is particularly significant, if or-COOH directly connect with carrier after haptens local macro ring of the feature structure vulnerable to carrier
The interference of border or steric hindrance influences the identification of body immune system, therefore, using 2- hydroxy-4-methyl benzene first as raw material, passes through
It is reacted with bromo- 2, the 2- dimethyl valeric acid of 5-, has synthesized the haptens with aldehyde radical, maximum possible remains original Gemfibrozil Capsules
Molecular structure is distributed the chemical structure of Gemfibrozil Capsules molecule with electronics and is barely affected, this has Gemfibrozil Capsules to obtain
The antibody of high degree of specificity provides guarantee.
The Gemfibrozil Capsules haptens synthesized in the present invention not only farthest remains the chemical structure of Gemfibrozil Capsules, but also
Linking arm with certain length goes immune animal, the potency of resulting antibody, spy with immunizing antigen prepared by this haptens
The opposite sex, affinity are all relatively good, and resulting antibody detects Gemfibrozil Capsules for ELISA method, and sensitivity is up to 1 μ g/L, with it
His cross reacting rate of Adjust-blood lipid class drug is low.
Detailed description of the invention
Fig. 1 Gemfibrozil Capsules hapten synthesis route map
Fig. 2 Gemfibrozil Capsules ELISA competition test curve graph
Specific embodiment
Present invention be described in more detail in the following with reference to the drawings and specific embodiments.Experiment as used in the following examples
Method is conventional method unless otherwise specified；Used material, reagent etc., unless otherwise specified, for commercially
Obtained reagent and material.
Embodiment 1: hapten synthesis
A kind of Gemfibrozil Capsules haptens, molecular structural formula are as follows:
It is used as the raw material of the antigen system of animal immune.
The preparation method of above-mentioned Gemfibrozil Capsules haptens is following (synthetic route is shown in attached drawing 1):
2- hydroxy-4-methyl benzaldehyde 0.5g is taken, acetonitrile 50mL is added to dissolve, clarification adds potassium carbonate 0.61g, adds 5- bromo- 2,
2- dimethyl valeric acid 0.84g, oil bath heating back flow reaction 3h；Stop reaction, be cooled to room temperature, rotate, removes acetonitrile, add water
50mL, ethyl acetate 100mL × 3, extraction three times, merge organic phase, upper silicagel column, with the ethyl acetate-pet of volume ratio 1:3
Gemfibrozil Capsules haptens can be obtained in ether chromatographic purifying.
Embodiment 2: artificial antigen synthesis and identification
A kind of Gemfibrozil Capsules artificial antigen, molecular structural formula made of the Gemfibrozil Capsules haptens described in embodiment 1 are as follows:
1, the synthesis of immunogene
The synthetic method of immunogene is as follows:
Gemfibrozil Capsules haptens 9.8mg is taken, adds ethyl alcohol 0.2mL to dissolve, obtains A liquid；Carrier protein 50mg is taken, is added
0.1mol/L sodium bicarbonate solution 3mL dissolution, obtains B liquid；A drop is added in B liquid, 4 DEG C are stirred for 24 hours, normal saline dialysis
3d is purified, is changed daily liquid 3 times, is dispensed, -20 DEG C save backup.
The identification of artificial antigen:
In the ratio of synthesis Gemfibrozil Capsules immunogene reaction haptens used, carrier protein and coupled product, carry out ultraviolet
(200~400nm) sweep measuring, by comparing three respectively the light absorption value of 260nm and 280nm calculate its combine than.Coupling
The maximum absorption band of object Gemfibrozil Capsules haptens-BSA with Gemfibrozil Capsules haptens, BSA maximum absorption band compared with have occurred it is bright
Aobvious variation shows that the synthesis of artificial antigen Gemfibrozil Capsules haptens-BSA is successful.It is computed, the knot of haptens and BSA
Composition and division in a proportion is 13:1.
2, the synthesis of coating antigen
9.8mg Gemfibrozil Capsules haptens is adjusted to 4.8mg, 50mg with the synthesis of immunogene by the synthetic method of coating antigen
Bovine serum albumin(BSA) replaces with 50mg ovalbumin (OVA).
The identification of artificial antigen:
In the ratio of synthesis Gemfibrozil Capsules coating antigen reaction haptens used, carrier protein and coupled product, carry out ultraviolet
(200~400nm) sweep measuring, by comparing three respectively the light absorption value of 260nm and 280nm calculate its combine than.Coupling
The maximum absorption band of object Gemfibrozil Capsules haptens-OVA with Gemfibrozil Capsules haptens, OVA maximum absorption band compared with have occurred it is bright
Aobvious variation shows that the synthesis of artificial antigen Gemfibrozil Capsules haptens-OVA is successful.It is computed, the knot of haptens and OVA
Composition and division in a proportion is 10:1.
Embodiment 3: monoclonal antibody preparation
A kind of Gemfibrozil Capsules specific antibody, be Gemfibrozil Capsules immunogen immune white mouse described in embodiment 2 is obtained,
The monoclonal immunoglobulin G that specific immune response can occur with Gemfibrozil Capsules, for detecting Gemfibrozil Capsules residual.
Above-mentioned Gemfibrozil Capsules monoclonal antibody the preparation method is as follows:
1) animal immune: immunogene is injected into Balb/c Mice Body, and immunizing dose is 150 μ g/, generates it anti-
Serum.
2) the Balb/c mouse boosting cell and myeloma cell SP20 for generating specific antibody cell fusion and cloning: are taken
Fusion measures cell supernatant using indirect competitive enzyme-linked immunosorbent analysis method, screens positive hole.Using limiting dilution assay to sun
Property hole carry out cloning, obtain and establish produce monoclonal antibody hybridoma cell strain.
3) cell cryopreservation and recovery: taking the hybridoma in logarithmic growth phase that cell suspension is made with frozen stock solution, point
Loaded on cryopreservation tube, saved for a long time in liquid nitrogen.Cryopreservation tube is taken out when recovery, is immediately placed in 37 DEG C of water-bath middling speeds and is melted, centrifugation removal
After frozen stock solution, culture culture in glassware is moved into.
4) preparation and purification of monoclonal antibody: using method is induced in vivo, Balb/c mouse (8 week old) Intraperitoneal injection is gone out
Bacterium paraffin oil is injected intraperitoneally hybridoma, acquires ascites after 7~10 days after 7~14 days.Through octanoic acid-saturated ammonium sulfate method into
The purifying of row ascites, purity is through SDS-PAGE electroresis appraisal, bottle packing, -20 DEG C of preservations.
The specific assay of antibody:
There are many antibody prepared by the immunogene (protein or polypeptide) of antigenic determinant for apparatus, wherein the antibody molecule contained
Often mixture.When there is two kinds of antigens of first, second, when there is the identical antigenic determinant in identical or part in molecular structure,
First antigen can be with the antibody response of second antigen, and second antigen can also be with the antibody response of first antigen, referred to as cross reaction.Antibody
Specificity just refers to the ability of its homospecificity antigen binding compared with the antigen-analogues ability.It is common to intersect instead
Answer activity as the major criterion of evaluation.Cross reaction is smaller, and the specificity of antibody is then better.
Specific antigen and the like is serially diluted, respectively with same Gemfibrozil Capsules haptens-BSA antibody,
Standard curve is made by production standard curve same method, and finds out dosage and the analog inhibition of inhibiting rate 50% on curve
Dosage when rate 50%.Then the cross reacting rate of each analog is calculated.
Cross reacting rate of the anti-Gemfibrozil Capsules haptens-BSA antibody to Gemfibrozil Capsules and each analog: Gemfibrozil Capsules are
100%, pravastatin sodium, Bezafibrate, Atorvastatin calcium, fluvastatin sodium, Clofibrate, mevastatin, Lovastatin,
The equal < 0.1% of Simvastatin.It can be seen that prepared antibody specificity is preferable.
Embodiment 4: the foundation and application of Gemfibrozil Capsules enzyme-linked immunosorbent assay for measuring
One, the basic principle of Gemfibrozil Capsules ELISA measuring method
It is using indirect competitive enzyme-linked immunosorbent analysis method, i.e., Gemfibrozil Capsules molecule and macromolecular carrier (such as protein) is even
Join compound obtained to be adsorbed in solid phase carrier (96 hole elisa Plates) as coating antigen, is prepared into solid phase antigen, is then added
Sample to be tested and corresponding antibodies.Gemfibrozil Capsules and antibody in solid phase antigen, sample to be tested are at war with association reaction, to test sample
Gemfibrozil Capsules content is more in product, then the antibody being bonded on solid phase antigen is few, otherwise combines the antibody on solid phase antigen more,
Then ELIAS secondary antibody is added, finally carries out colour developing with substrate and is measured.When lucky in one timing of amount of antibody, the sample to be tested of addition
Fei Luoqi amount is more, and the antibody in conjunction with solid phase antigen is fewer, and the ELIAS secondary antibody in conjunction with antibody is also fewer, and color reaction is just
Weaken, conversely, then color reaction enhances, thus can be according to the standard curve of known Gemfibrozil Capsules amount and the absorbance of sample to be tested
Value, extrapolates the concentration of Gemfibrozil Capsules in sample to be tested.
Two, component involved in Gemfibrozil Capsules ELISA measuring method
(1) it is coated with the ELISA Plate of Gemfibrozil Capsules artificial antigen (Gemfibrozil Capsules haptens-ovalbumin conjugate)；
(2) Gemfibrozil Capsules serial standards solution: concentration is respectively 0 μ g/L, 1 μ g/L, 3 μ g/L, 9 μ g/L, 27 μ g/L, 81
μg/L；
(3) antibody working solution: Gemfibrozil Capsules monoclonal antibody working solution described in embodiment 3；
(4) ELIAS secondary antibody: with the sheep anti mouse antiantibody of horseradish peroxidase-labeled；
(5) substrate developing solution: being made of A liquid and B liquid, and A liquid is the aqueous solution of 2% urea peroxide, and B liquid is 1% tetramethyl
The aqueous solution of benzidine；
(6) terminate liquid: 2mol/L aqueous sulfuric acid；
(7) cleaning solution: contain 1.0% Tween-20,0.02 ‰ sodium azide, the phosphate of pH value 7.4,0.05mol/L
Buffer；
(8) it redissolves liquid: containing 5% bovine serum albumin(BSA), the phosphate buffer of pH value 7.4,0.02mol/L.
Three, in Gemfibrozil Capsules ELISA measuring method each component preparation
1, it is coated with the preparation of the ELISA Plate of Gemfibrozil Capsules artificial antigen (Gemfibrozil Capsules haptens-ovalbumin conjugate)
Gemfibrozil Capsules coating antigen described in embodiment 2 is diluted to 0.2 μ g/mL with coating buffer, is coated with 96 hole polyphenyl
Ethylene ELISA Plate, 100 μ L of every hole, 37 DEG C of incubation 2h, coating buffer of inclining are washed 3 times with cleaning solution, and each 30s is patted dry, then
Be added 200 μ L confining liquids in every hole, 37 DEG C of incubations 2h, liquid in hole of inclining, it is dry after with the preservation of aluminium film vacuum sealing.
Coating buffer used and confining liquid are as follows:
It is coated with buffer: the carbonate buffer solution of pH9.6,0.05mol/L；
Confining liquid: contain 0.5% horse serum, 0.1% sodium azide, 3% casein, pH value 7.2,0.02mol/L
Phosphate buffer.
2, the preparation of Gemfibrozil Capsules serial standards solution
It is respectively 0 μ g/L, 1 μ g/L, 3 μ g/L, 9 μ g/ that Gemfibrozil Capsules standard items, which are diluted to concentration, with standard dilutions
L,27μg/L,81μg/L；Standard dilutions are to contain 5% bovine serum albumin(BSA), the phosphate of pH value 7.4,0.02mol/L
Buffer.
3, the preparation of antibody working solution
Gemfibrozil Capsules monoclonal antibody described in embodiment 3 is diluted 1000 times with antibody diluent, obtains antibody work
Make liquid；Antibody diluent is the phosphate buffer containing 2.5% casein and 0.03 ‰ sodium azide.
4, the preparation of ELIAS secondary antibody
(1) preparation of sheep anti mouse antiantibody
Using sheep as immune animal, pathogen-free domestic sheep is immunized using source of mouse antibody as immunogene, it is anti-to obtain sheep anti mouse
Antibody.
(2) preparation of ELIAS secondary antibody
Sheep anti mouse antiantibody is coupled with horseradish peroxidase using Over-voltage protection, it is then dilute with ELIAS secondary antibody
It releases liquid and is diluted 500 times；The ELIAS secondary antibody dilution is containing 0.5% bovine serum albumin(BSA), pH value 7.4,0.02mol/L
Phosphate buffer.
Four, with remaining Gemfibrozil Capsules in above-mentioned Gemfibrozil Capsules ELISA measuring method test sample
(1) sample pre-treatments
Water sample is subjected to 2 times of dilutions with liquid is redissolved.
(2) it detects
Serial standards solution is added to being coated in Gemfibrozil Capsules haptens-ovalbumin conjugate micropore of enzyme marker plate
Or 50 μ L of sample solution, 50 μ L of ELIAS secondary antibody is added immediately, adds 50 μ L of monoclonal antibody working solution, gently oscillation mixes,
30min is reacted in cover board membrane cover plate 25 DEG C of light protected environments of postposition；Liquid in hole is poured out, cleaning solution 250 μ L, 10s is added in every hole
After pour out liquid in hole, repeat operation total board-washing 5 times, patted dry with blotting paper；Substrate developing solution A liquid and B liquid is added in every hole
Each 50 μ L, gently oscillation mixes, and reacts 15min in cover board membrane cover plate 25 DEG C of light protected environments of postposition；50 μ of terminate liquid is added in every hole
L, gently oscillation mixes, and sets microplate reader at 450nm, measures the absorbance value in every hole.
(3) Analysis of test results
With the absorbance values (B) of the standard solution of each concentration obtained divided by first standard solution (0
Standard) absorbance values (B0) multiplied by 100%, obtain percentage absorbance value.
Using the logarithm of Gemfibrozil Capsules standard concentration as X-axis, percentage absorbance value is Y-axis, draws canonical plotting (figure
2).The percentage absorbance value of sample solution is calculated with same method, the concentration of each corresponding sample then can be from standard song
The residual quantity of Gemfibrozil Capsules in sample is read on line.The analysis of testing result can also use regression equation method, meter in the present invention
Calculate sample solution concentration.The analysis of testing result can also utilize computer professional software in the present invention, this method is more convenient for greatly
The quick analysis of sample is measured, entire detection process only needs 1.0h that can complete.
Five, above-mentioned Gemfibrozil Capsules ELISA measuring method detection effect evaluation
(1) minimum detection limit
20 parts of negative water sample without Gemfibrozil Capsules are taken, will be surveyed after sample progress pre-treatment with above-mentioned Gemfibrozil Capsules ELISA
Determine method to detect it, detection limit is indicated plus 3 times of standard deviations with the average value of 20 parts of sample detection concentration.
The result shows that this method is limited to 2 μ g/L to the lowest detection of water sample.
(2) accuracy and precision
Using the rate of recovery as accuracy estimating index, the testing result relative standard deviation of a certain concentration samples of replication
(RSD%) it is used as precision evaluation index.Calculation formula are as follows: the rate of recovery (%)=actual measured value/theoretical value × 100%,
Middle theoretical value is the addition concentration of sample；Relative standard deviation RSD%=SD/X × 100%, wherein SD is standard deviation, and X is
The average value of determination data.
Gemfibrozil Capsules are added respectively into the water sample without Gemfibrozil Capsules, make its final concentration of 2 μ g/L, 4 μ g/L, 8 μ g/L,
It will be detected with above-mentioned Gemfibrozil Capsules ELISA measuring method after sample progress pre-treatment after addition, selects 3 batches of examinations
Agent, each concentration do 5 it is parallel, calculate separately the rate of recovery and batch in, batch between RSD, the results are shown in Table 1.The result shows that water sample
Average recovery rate 80%~100%, in batch, batch between RSD within 10%.
1 accuracy of table and precision experiment result
(3) storage life
Involved main agents are finally provided in the form of working solution in above-mentioned Gemfibrozil Capsules ELISA measuring method,
It is assembled into kit, greatly reduces the error of liquid relief and operation, while small in size, is easy to carry about with one and is transported to terminal client
Place is more suitably applied to execute-in-place detection and high-volume pattern detection, also saves express transportation cost.
Kit preservation condition is 2-8 DEG C, by measurement in 12 months, the maximum absorbance value (zero standard) of kit,
50% inhibition concentration, Gemfibrozil Capsules TIANZHU XINGNAO Capsul are in the normal range.In view of transport and use process in have it is non-just
Normal preservation condition occurs, and kit is placed to 8 days progress accelerated aging tests at 37 DEG C, as a result the indices of the kit
Comply fully with requirement；In view of kit freezing happens, kit is placed 8 days in -20 DEG C of refrigerators, as a result the reagent
The indices of box are also completely normal.It can be obtained from result above, kit can at least save 12 months at 2-8 DEG C or more.
Claims (7)
1. a kind of Gemfibrozil Capsules haptens, it is characterised in that its molecular structural formula are as follows:
2. the preparation method of Gemfibrozil Capsules haptens described in a kind of claim 1, it is characterised in that include the following steps:
2- hydroxy-4-methyl benzaldehyde 0.5g is taken, acetonitrile 50mL is added to dissolve, clarification adds potassium carbonate 0.61g, adds bromo- 2, the 2- bis- of 5-
Methylvaleric acid 0.84g, oil bath heating back flow reaction 3h；Stop reaction, be cooled to room temperature, rotate, removes acetonitrile, add water 50mL,
Ethyl acetate 100mL × 3, extraction three times, merge organic phase, upper silicagel column, with the ethyl acetate-light petrol layer of volume ratio 1:3
Analysis purifying, can be obtained Gemfibrozil Capsules haptens.
3. a kind of Gemfibrozil Capsules artificial antigen, it is characterised in that its molecular structural formula are as follows:
The carrier protein is bovine serum albumin(BSA).
4. the preparation method of Gemfibrozil Capsules artificial antigen described in a kind of claim 3, it is characterised in that include the following steps:
Gemfibrozil Capsules haptens 9.8mg is taken, adds ethyl alcohol 0.2mL to dissolve, obtains A liquid；Carrier protein 50mg is taken, 0.1mol/L carbon is added
Sour hydrogen sodium solution 3mL dissolution, obtains B liquid；A drop is added in B liquid, for 24 hours, normal saline dialysis purifies 3d, daily for 4 DEG C of stirrings
It changes liquid 3 times, dispenses, -20 DEG C save backup.
5. a kind of application of the Gemfibrozil Capsules haptens described in claim 1 in terms of the antigen system for preparing animal immune, special
Sign is that the raw material of the antigen system as animal immune.
6. Gemfibrozil Capsules artificial antigen described in a kind of use claim 3 is immune, white mouse is obtained, can occur with Gemfibrozil Capsules
The Gemfibrozil Capsules monoclonal antibody of specific immune response.
7. a kind of application of the Gemfibrozil Capsules monoclonal antibody described in claim 6 in detection Gemfibrozil Capsules residual.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810991947.1A CN109232238B (en) | 2018-08-29 | 2018-08-29 | Gemfibrozil hapten, artificial antigen and antibody, and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810991947.1A CN109232238B (en) | 2018-08-29 | 2018-08-29 | Gemfibrozil hapten, artificial antigen and antibody, and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109232238A true CN109232238A (en) | 2019-01-18 |
| CN109232238B CN109232238B (en) | 2022-05-17 |
Family
ID=65069360
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810991947.1A Active CN109232238B (en) | 2018-08-29 | 2018-08-29 | Gemfibrozil hapten, artificial antigen and antibody, and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109232238B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109970550A (en) * | 2019-03-26 | 2019-07-05 | 深圳市易瑞生物技术股份有限公司 | Sorbic acid haptens, artificial antigen, antibody and its synthetic method and application |
| CN113563181A (en) * | 2021-08-11 | 2021-10-29 | 浙江精进药业有限公司 | Method for removing gemfibrozil impurities |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1326358A (en) * | 1998-08-26 | 2001-12-12 | 葛兰素集团有限公司 | Method of DNA vaccination |
-
2018
- 2018-08-29 CN CN201810991947.1A patent/CN109232238B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1326358A (en) * | 1998-08-26 | 2001-12-12 | 葛兰素集团有限公司 | Method of DNA vaccination |
Non-Patent Citations (4)
| Title |
|---|
| M. CERMOLA ET AL.,: ""Phototransformation of fibrate drugs in aqueous media"", 《ENVIRON CHEM LETT》 * |
| SEDA DEMIRELTOPEL ET AL.,: ""Synthesis and characterization of Bodipy functionalized magnetic iron oxide nanoparticles for potential bioimaging applications"", 《COLLOIDS AND SURFACES B: BIOINTERFACES》 * |
| 刘丽主编: "《胶体金免疫层析技术》", 30 September 2017, 河南科学技术出版社 * |
| 贾连群，雷萍主编: "《现代基础医学理论与技术进展》", 31 August 2015, 中国医药科技出版社 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109970550A (en) * | 2019-03-26 | 2019-07-05 | 深圳市易瑞生物技术股份有限公司 | Sorbic acid haptens, artificial antigen, antibody and its synthetic method and application |
| CN109970550B (en) * | 2019-03-26 | 2021-09-07 | 深圳市易瑞生物技术股份有限公司 | Sorbic acid hapten, artificial antigen, antibody, synthetic method and application thereof |
| CN113563181A (en) * | 2021-08-11 | 2021-10-29 | 浙江精进药业有限公司 | Method for removing gemfibrozil impurities |
| CN113563181B (en) * | 2021-08-11 | 2024-04-26 | 浙江精进药业有限公司 | Method for removing gemfibrozil Ji Zazhi |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109232238B (en) | 2022-05-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101571539B (en) | Elisa kit for detecting cephalo-type medicine and application thereof | |
| CN102967709B (en) | Detect enzyme linked immunological kit and the application thereof of zearalenone medicine | |
| CN101256188A (en) | ELISA kit for detecting lincomycin medicine as well as usage thereof | |
| CN106771137B (en) | Detect enzyme linked immunological kit and its application of Nicarbazin | |
| CN101776685B (en) | Enzyme linked immunosorbent assay kit for detecting trimethoprim medicament and application thereof | |
| CN105481859A (en) | Artificial antigen of tadalafil and analogues thereof, antibody and ELISA kit thereof | |
| CN102080067B (en) | Method for detecting deoxynivalenol and special reagent kit thereof | |
| CN109232238A (en) | Gemfibrozil Capsules haptens, artificial antigen and antibody and its preparation method and application | |
| CN109369435A (en) | Tolfenamic Acid haptens, artificial antigen and antibody and its preparation method and application | |
| CN101907623B (en) | Method for detecting gentamicin and/or micronomicin and special quantum dot fluorescence immunoassay kit thereof | |
| Zhang et al. | Rapid monitoring of dipropyl phthalate in food samples using a chemiluminescent enzyme immunoassay | |
| CN105785012A (en) | Enzyme linked immunosorbent assay kit for detecting ribavirin and application thereof | |
| CN101936985A (en) | A method for detecting diethylstilbestrol and its special chemiluminescence immunoassay kit | |
| CN103364553A (en) | Enzyme linked immunosorbent assay kit for detecting nitroimidazole drugs and application thereof | |
| CN100492009C (en) | ELISA kit for detecting fluoromethylquinoline and detection method thereof | |
| CN106872681B (en) | Amikacin and the two-in-one quick detection enzyme linked immunological kit of kanamycins and its application | |
| CN101955537A (en) | Anti-ractopamine antibody and application thereof | |
| CN105158472A (en) | Enzyme linked immunosorbent assay kit for detecting metalaxyl residues and using method | |
| CN109061168A (en) | Detect enzyme linked immunological kit and its application of diclazuril | |
| CN109813880A (en) | A kind of remaining enzyme linked immunological kit of detection Trimethoprim | |
| CN109180507A (en) | Ketoprofen haptens, artificial antigen and antibody and its preparation method and application | |
| CN1308686C (en) | Kit for detecting chloromycetin | |
| CN103508910B (en) | Beta-stimulants class haptens and its preparation method and application | |
| CN112142756A (en) | Preparation of aflatoxin M1 hapten and holoantigen as well as preparation and application of rapid transfer quantitative kit | |
| CN109536457A (en) | A kind of hybridoma cell strain that secreting anti-imidacloprid monoclonal antibody and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |